Fundamental Gas Chromatograph Ratimarth Bunlorm ratimarth@ - - PowerPoint PPT Presentation

Ratimarth Bunlorm ratimarth@ scispec.co.th

Fundamental Gas Chromatograph

Chromatography

• Chromatography : Analytical technique that depends on separation of

components in sample

• Sample components are separated and detected
• Separation : Between two phases

– Stationary Phase – Mobile phase

Gas Chromatography

• Gas Chromatography (GC) : Chromatography technique which gas is

used as mobile phase

• Sample will be injected into the system, Injection port where all

components are vaporized and swept into the column

• Sample components will then be separated according to the interaction

with stationary phase and eluted to detector.

Column Carrier Gas Detector

GC System Components

Detector Injector Column Oven Carrier gas Detector Gas Cylinder

GC System Components : Carrier gas

Detector Injector Column Oven Carrier gas Detector Gas Cylinder

Carrier Gas Selection : Gas Purity (impurity)

• Impurities can alter stationary phase in column and cause high

background (noise), contamination

– Free from moisture, organic hydrocarbons and oxygen – Free from components those associate or interfere the analysis – Recommended at least 99.995% – Purified traps must be installed

Carrier Gas Selection : Speed of Analysis & Resolution

• Speed of analysis : The lighter carrier gas, the faster analysis time.

– With the same resolution (separation performance), Helium provides shorter

analysis time than Nitrogen

– Helium is lighter than Nitrogen so it travels through column faster than Nitrogen – At the same supplied pressure, Helium has more density than Nitrogen so Helium

will provide better peak shape (resolution).

He N2

Carrier Gas & Speed of Analysis

Average Linear Gas Velocity , cm/sec HETP, mm Nitrogen Helium Hydrogen

GC System Components : Injector

Detector Injector Column Oven Carrier gas Detector Gas Cylinder

Injector

• Injector : The area in which the sample is introduced, evaporated

instantaneously & carried to the column with a minimum of band spreading.

• Concerned parameters :

– Sample size – Temperature – Carrier gas pressure/flow control

Types of Injection

• Packed Column Injector
• Split/Splitless Injector (Capillary Injector)
• On-Column Injector

– Packed – Capillary – Cold On-Column

• PTV : Pressure Temperature Vaporizing Injector
• Injection Valve

– Gas Sampling Valve (GSV) – Liquid Sampling Valve (LSV)

• Can be used for

– Capillary column 0.1, 0.25, 0.32

mm ID

– Wide bore column (0.53 mm.ID) – Packed column (requires

conversion kit)

• Can be operated in two modes

– Split – Splitless

Split/Splitless Injector

• Split Injection

– Only a part of the sample transfers

into the column. The rest discharges through the split vent

– The ratio of the split flow to the col

umn flow so called “split ratio” determines the amount

• f sample that enter the column

Split injection technique

• Splitless injection is suitable for

– The analysis of compounds present in

very low concentration with relatively dirty matrices.

– Allows a portion of entire sample to

enter the column without splitting

– Split vent is closed during sample inje

ction and transfer to the column, Once the transfer is over, the split vent is reopened to flush the vaporizing chamber for any remaining sample vapors.

Splitless injection technique

Injector : general maintenance for user

• Monitor contamination
• Set optimum injection temperature (provide complete sample

vaporization)

• Inject clean sample, appropriate sample size
• Clean liner, Change liner
• Change liner seal or liner o-ring
• Change septum

GC System Components : Column and Oven

Detector Injector Column Oven Carrier gas Detector Gas Cylinder

Selection of stationary phase

• The rule :

– A non-polar component is dissolved in a non-polar liquid phases – A polar component is dissolved in a polar liquid phase.

• Elution Order of interested components vs. matrix
• Resolution : Separation Capability
• Temperature limitation of the stationary phase

Column Oven

• Provides a stable heating environment for the analytical column.
• Must heats and cools quickly with efficient air circulation to

ensures a high degree of thermal stability

Oven Temperature vs. Resolution

• Components in the sample will be separated under optimum column

temperature

• Increases oven temperature trend to reduce in resolution
• Ultimate Goal is “all components are separated with the shortest analysis

time”

Isothermal 70 C

GC System Components : Detector

Detector Injector Column Oven Carrier gas Detector Gas Cylinder

§ Miniaturized instant connect detectors

• Available: FID, ECD, TCD, NPD, PDD and FPD (also dual flame)
• Single bodies including cells, heater and gas feeding
• Reduced volumes for increased sensitivity
• Up to four can be mounted and operated at the same time
• Fast acquisition speed: up to 300 Hz
• Enhanced Linearity
• Easy access to removable parts for maintenance

§ Front-end to Mass Spectrometers for increased

selectivity and sensitivity

Robust and Reliable Performance

TCD FID ECD NPD PDD

TRACE 1300 Series GC “Detector” modules

Fundamental of Mass Spectrometer

Why GC/MS?

• Universal and specific

–

Full scan for unknown sample

–

SIM, MIM for specific (interested) mass

• High Sensitivity

–

ppt level

• Provides identification with standard or library spectrum
• Interference-free quantitation (SIM or MIM)
• Isotopic information
• Confirmation of other conventional detectors

What is Mass Spectrometry?

• The production of ions that are subsequently separated or

filtered according to their mass-to-charge (m/z) ratio and detected.

• The resulting mass spectrum is a plot of the (relative) abundance
• f the produced ions as a function of the m/z ratio.”

What is “Mass Spectrum” ?

• Graph of Relative Ion Intensity vs. m/z
• Ion Fragments detail structure and molecular weight of compound

CCl3

CCl2 CCl Cl

Ion Abundance Other are called “fragments” “parent mass” CCl4 MW=152

Mass Spectrum

p,p'-DDT MW: 352 Benzene MW: 78 Dodecane MW: 170 Amphetamine MW: 135

Total Ion Chromatogram (TIC), Extracted Ion Chromatogram (EIC), and Mass Spectrum

TIC EIC mass 303 Spectrum peak at RT 2.56 min Full scan 35-450 amu

Components in GC/MS

GC Ion source Mass Analyzer

Detector

Turbomolecular Pump Fore Pump Data System MS Electronics

Transfer line

Fore Vacuum Gauge Ion Gauge

Transfer line

• “Bridge” between GC and MS’s Ion Source
• Vacuum tube with have heater coil on the internal tube.
• GC column is inserted inside the internal tube.
• High temperature (200-350 C) is set to protect sample condensation.
• Type

– Direct capillary transfer line (most widely used) – GC column connect directly to

ion source

– Open/Split transfer line – Splitter transfer line – Jet separator

Ion source

• Ion Source covert sample molecules (neutral) into charged molecules or

molecular ions.

• Charged molecules (Molecular ions) can be easily manipulated with

electrical and magnetic fields

• Process in mass spectrometer are using DC, RF to

– Focusing : arrange the molecular ion to travel in a straight direction – Diverting : turn the direction of molecular ion – Filtering : get rid of unwanted molecular ion – Detecting : detect those interested molecular ion

Ion Source Cartridge (iSQ)

Ion Cartridge Sleeve RF Lens/Lens 3 Lens 2 Lens 1 Ion Volume Repeller Ion Volume/ Repeller Insulator Repeller Nut Repeller Spring Locking Ring Ion Cartridge Sleeve RF Lens/Lens 3 Lens 2 Lens 1 Ion Volume Repeller Ion Volume/ Repeller Insulator Repeller Nut Locking Ring

Ionization Methods in GCMS

• Electron Ionization
• Chemical Ionization

– Positive Ion Chemical Ionization – Negative Ion Chemical Ionization

Electron Ionization

Ion Repeller Transfer line from GC Filament Electron Beam Focusing Lens Molecular Ions

PCI : Positive Ion Chemical Ionization

• Reagent gas reacts with electrons to form primary ions
• Primary ions react with CH4 and form collided ions
• Collided ions react with sample molecules (soft ionization) and form

molecular ions

• Molecular ions present in form of [M+H]+, [M-H]+, [M+17]+,[M+29]+,

[M+41]+

• Main use is molecular weight confirmation (clean spectra)
• Example of reagent gas : CH4, Isobutane

Adduct Formation in PICI

M-1

EI versus PCI for Pesticides (heptachlor MW 336)

EI Spectrum of Heptachlor Intensity is low for any single m/z ion. PICI Spectrum of Heptachlor Intensity is concentrated in [M+H]+ ion. Spectrum is simpler.

In PICI, sample is not fragmented. Therefore, PICI will provide higher ion intensity Which means better detection limit when compares with EI

Ion Transmission

• Lens :

– Applying appropriate voltage to lens can be used to induced molecular ions into

certain distance and direction

• Multi-pole rods :

–

quadrupoles , hexapoles, octapoles are widely used to transmit ions for longer distance

Mass Analyzers

1. Quadrupole or Single Quadrupole 2. Triple Quadrupole 3. Time of Flight (TOF) 4. Magnetic Sector 5. Orbitrap

Single Quadrupole Mass Analyzer

Quadrupole - consists of two sets on opposing

• rods. This mass analyzer uses a combination
• f RF(AC) and DC modulation to sort ions. This

analyzer provides nominal mass resolution

Quadupole Mass Filter Operation

+20

• 20
• 20

+20

At Time 0

m/z= 4+ m/z= 100+ m/z= 500+

Quadupole Mass Filter Operation

+140

• 140
• 140

+140 At Time 1 m/z= 4+ m/z= 100+ m/z= 500+

Quadupole Mass Filter Operation

m/z= 4+ m/z= 100+ m/z= 500+ At Time = 2 +100

• 100

+100

• 100

Quadupole Mass Filter Operation

m/z= 4+ m/z= 100+ m/z= 500+ At Time = 3

• 140

+140

• 140

+140

Quadupole Mass Filter Operation

m/z= 4+ m/z= 100+ m/z= 500+ At Time = 4 +140

• 140

+140

• 140

ISQ 7000 GCMS – Designed with Intention

• Full Scan

– Set a mass range to cover sample’s molecular ions – Get spectrum for identification – Good for unknown but Low sensitivity

• Selected Ion Monitoring (SIM)

– Select one or a few molecular ions those will be monitored – Lost spectrum information – High sensitivity but may cause false positive error

Operation modes in Single Quad MS

Triple Quadrupole Mass Analyzer

• Triple Quadrupole - consists of two sets of quadrupole with one collision cell in
• between. This mass analyzer uses a combination of RF and DC modulation to sort ions

just like single quadrupole. Q1 and Q3 work like mass filter (using RF and DC) while Q2 works as a Collision cell (RF only and Collided gas). Q1 can selected any precursor (parent mass) and pass it into collision cell (Q2) where precursor are fragmented and pass through Q3 for ion sorting again. This analyzer provides high sensitivity with fast confirmation analysis.

Selected Reaction Monitoring (SRM or MRM)

Quantitation of target compounds in matrix samples

Q1 selects the precursor ion Q3 selects the product ion

Argon Collision Gas

Select Fragment Detect

(mainlib) P arathion 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 50 100 15 29 39 45 65 75 81 97 109 125 139 150 155 172 186 201 218 235 246 263 275 291 N O O O P O S O

Structure Specific Selectivity by QQQ : Parathion-Ethyl

M+ m/z 291,03

SRM Precursor Ion

(used for SIM in single quad methods)

SRM Product Ion m/z 97,01 m/z 109,01

NIST Library Spectrum

Full scan/SRM Acquisition

• Full scan
• SRM
• Spectra from

FS/SRM Method

• NIST Spectra

Detector : Dynode Electron Multiplier

• Dynode converses Molecular ions into electron

– Continuous Dynode – Discrete Dynode

• Electron are then sent to multiplier for signal

enhancing

Photo courtesy from SGE & ETP, Wikipedia

Off –axis dynode and EM

• Off axis dynode

– High voltage is applied (+/-10 KV) for

high signal (accelerate ion velocity from mass analyzer to dynode)

– Induces only molecular ions to hit

dynode

• Electrons from dynode hit internal wall
• f EM.
• Multiplication process amplifies for

more signals Dynode

Electron Multiplier

Pumps

• High Vacuum Pumps (10-3 to 10-10 Torr)

– Oil Diffusion

• No moving parts

– Turbomolecular

• Clean - no oil
• Mechanical Backing Pump, (Fore Pump) (atm. to 10-3 Torr)

– Rotary vane

Step 1. Insert removal tool Step 2. Remove source Step 3. Hot source is held in tool Step 4. Push source out of tool

User maintenance :Vacuum probe interlock

User maintenance :Vacuum probe interlock

ISQ 7000 Series…

ISQ 7000 NeverVent EI & CI ISQ 7000 AEI Affordable first entry Accessible high performance High-throughput solution High-throughput solution Ultra high sensitivity and robustness

Perfect for today, ready for tomorrow

• Fit for purpose GC-MS solution
• Grows with evolving regulatory requirements
• Base to advanced configurations
• Full field upgrade path

66L/s ExtractaBrite 300L/s ExtractaBrite ISQ 7000 NeverVent EI

240L/s ExtractaBrite 300L/s ExtractaBrite TSQ 9000 NeverVent EI TSQ 9000 NeverVent EI & CI TSQ 9000 AEI Most accesible entry from SQ>TQ Affordable performance High-throughput solution High-throughput solution Ultra high performance and robustness

Perfect for today, ready for tomorrow

• Grows with laboratory requirements
• Base to advanced configurations
• Full field upgrade path

ISQ 9000 Series…

Application…...

www.scispec.co.th

Scispec website : Application…...

GC and GCMS application support.

Application : Biodiesel

• Free and Total glycerin : ASTM D6584 / EN 14150
• Total FAME and Linolenic Acid Methyl Ester : EN 14103
• Methanol Content : EN 14110

Total FAME and Linolenic Acid Methyl Ester : EN 14103

The cetane namber of biodiesel depends on the distribution of fatty acids in the original oil. Thus a reliable characterization of FAME is essential for a more accurate calculation of the cetane index. EN 14103 is a standard method for determination of esters and linolenic acid methyl ester and can be applied to biodiesel analysis. EN 14103 requires GC analysis.

Total FAME and Linolenic Acid Methyl Ester : EN 14103

By incorporating the backflush option into the PTV injector, heavy compounds can be vented out of the inlet system, effectively preventing column contamination while still allowing efficient transfer of compounds of interest.

Methanol Content : EN 14110

Methanol in B100 is a matter of safety since even small amounts of this material can reduce the flash point of the biodiesel. Moreover, residual methanol can affect fuel pumps, seals and elastomers and can result in poor combustion properties. EN 14110 requires a headspace GC method, based on either polar or non-polar columns, and is applicable for a concentration range from 0.01% m/m to 0.5% m/m of methanol (MeOH).

Multi-Residue Pesticide Analysis in Herbal Products Using Accelerated Solvent Extraction with a Triple Quadrupole GC-MS/MS System

Sample Preparation

Multi-Residue Pesticide Analysis in Herbal Products Using Accelerated Solvent Extraction with a Triple Quadrupole GC-MS/MS System

Dried leaves , fruits or seeds and other herbal products Weight 10 g of sample. Mixed with DE and load into the extraction cells. Concentrated Sample and injection with GC

GC : Condition MS/MS : Condition

Multi-Residue Pesticide Analysis in Herbal Products Using Accelerated Solvent Extraction with a Triple Quadrupole GC-MS/MS System

SRM : More than 80 compound

Calibration and Detection limit.

Calibration level : 0.004 µg/mL to 1.0 µg/mL(This range represents an analyte concentration of 0.01 to 2.5 mg/kg in the samples) Sensitivity (LOD) Terbacill Alachor Tolyfluanid Pyridaben

Sample Result…..

Application note 52291

PY-GCMS

Pyrolyzer

Information from polymeric Materials by Heating

Pyrolyzer

Pyrolysis of Polymeric materials and pyrolyzates

Pyrolyzer

Typical pyrogram of polyethylene at 600ºC

Typical pyrograms

• A: Identification of polymeric materials
• Unknown materials (PP/ PVC/ SBR?)
• B: Structural characterization of polymers
• C: Mechanisms and kinetics of polymer degradation
• stereo regularity
• C=C-C-C*-C-C*-C-C
• C
• C
• C
• C
• [
• ]
• D: Qualitative and quantitative analysis of additives
• Various monomers
• chain-end
• MW / Sequence distributions (x-n-m-n..)
• Blend or copolymer (X+Y or X&Y)
• X • [ CH2CH=CHCH2] [CH2CH(CN)] [ CH2CH(C6H5)]•]
• x•[
• n
• m •y
• X

Characterization of Polymers by PY-GC/MS

77

สภาวะเครื่อง GCMS

• Injector

– Temperature 300 oC – Split 200:1 – Carrier gas flow 1.0 ml/min

• Oven

– Initial 70 oC hold 1min ramp 1 ; 10

• C/min to 320 oC hold 8 min.
• MS

– Temperature 250 oC – Scan 35-550 amu. สภาวะเครื่อง Pyrolyzer

• Single-Shot Analysis
• Furnace Temperature 600 oC
• Interface Temperature 300 oC

µÑÇ Í Âè Ò §¡ Ò ÃÇÔ à ¤ ÃÒ ÐËì ´ é Ç PY-GCMS

• Sample cup

• Step 1
• Knife
• GC
• MS
• Pyrolyzer
• Sample cup
• Step 3
• Place a sample
• in the sample cup
• No solvent extraction
• Step 2
• 0.1- 0.5mg

ขั้นตอนการเตรียมตัวอย่าง

ผลการวิเคราะห์ตัวอย่างที่ 1

0.1 5.0 10.0 15.0 20.0 25.0 30.0 31.1 Time [min]

• 1.0e8

0.0e0 1.0e8 2.0e8 3.0e8 4.0e8 5.0e8 6.0e8 7.0e8 8.0e8 9.0e8 Intensity [counts]

RT: 3.20 RT: 4.11 RT: 4.50 RT: 5.16 RT: 5.62 RT: 6.49 RT: 12.84 RT: 12.92 RT: 13.31 RT: 14.05 RT: 14.12 RT: 14.47 RT: 14.96 RT: 15.13 RT: 15.25 RT: 15.36 RT: 15.42 RT: 15.56 RT: 15.68 RT: 15.81 RT: 15.85 RT: 15.97 RT: 16.09 RT: 16.28 RT: 16.33 RT: 16.41 RT: 16.51 RT: 16.67 RT: 17.01 RT: 17.09 RT: 17.22 RT: 17.41 RT: 17.48 RT: 17.77 RT: 17.84 RT: 18.03 RT: 18.25 RT: 19.18 RT: 20.64 RT: 20.85 RT: 21.06 RT: 21.25 RT: 21.74 RT: 22.17 RT: 22.28 RT: 22.80 RT: 22.95 RT: 23.05 RT: 23.46 RT: 23.66 RT: 23.85 RT: 24.25 RT: 24.29 RT: 24.64 RT: 27.18 RT: 27.29 RT: 27.35

min counts

PY-DSS_170712 #11 GPPS_PY_2 TIC TIC

• Toluene
• Styrene
• Methyl styrene
• EMDP
• (2,3-diphenylcyclopropyl) methyl phenyl sulfoxide ,trans
• Ethyl benzene

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• Rank.2 : Styrene-butadiene copolymer ABA block, 85% styrene (C1-C40) Qual. 85
• Rank.3 : Acrylonitrile-Butadiene-Styrene copolymer ; ABS (C1-C40) Qual.84

ผลการวิเคราะห์ตัวอย่างที่ 2

0.1 5.0 10.0 15.0 20.0 25.0 30.0 31.1 Time [min]

• 1.0e8

0.0e0 1.0e8 2.0e8 3.0e8 4.0e8 5.0e8 6.0e8 7.0e8 8.0e8 Intensity [counts]

RT: 2.04 RT: 3.23 RT: 3.83 RT: 4.13 RT: 4.50 RT: 5.18 RT: 5.63 RT: 6.21 RT: 6.43 RT: 6.50 RT: 6.62 RT: 9.00 RT: 11.79 RT: 12.84 RT: 12.92 RT: 13.31 RT: 14.05 RT: 14.47 RT: 14.66 RT: 14.96 RT: 15.13 RT: 15.25 RT: 15.36 RT: 15.43 RT: 15.56 RT: 15.68 RT: 15.85 RT: 15.97 RT: 16.09 RT: 16.28 RT: 16.33 RT: 16.41 RT: 16.51 RT: 17.01RT: 17.09 RT: 17.22 RT: 17.41 RT: 17.48 RT: 17.77 RT: 17.84 RT: 17.96 RT: 18.03 RT: 18.25 RT: 19.18 RT: 19.77 RT: 20.85 RT: 21.06 RT: 21.25 RT: 22.17 RT: 22.28 RT: 22.95 RT: 23.05 RT: 23.46 RT: 23.86 RT: 24.25 RT: 24.29 RT: 27.18 RT: 27.28

min counts

PY-DSS_170712 #12 HIPS_PY_2 TIC TIC

• Styrene
• 1,3 - butadiene
• Toluene
• Ethyl benzene
• Methyl styrene
• EMDP
• (2,3-diphenylcyclopropyl) methyl phenyl sulfoxide ,trans

¼Å¡ Ò ÃÇÔ à ¤ ÃÒ ÐËì à Á× Í à · Õ Âº ¡ Ñ º ° Ò ¹ ¢é Í ÁÙ Å´ é Ò ¹ ¾Í ÁÔ à ÁÍ Ã ì ¼ è Ò ¹ «Í ¿ áÇà ì F-Search

• Rank.2 : Acrylonitrile-Butadiene-Styrene copolymer ; ABS (C1-C40) Qual.86
• Rank.3 : Styrene-butadiene copolymer ABA block, 85% styrene (C1-C40) Qual. 86

ผลการวิเคราะห์ตัวอย่างที่ 3

0.1 5.0 10.0 15.0 20.0 25.0 30.0 31.1 Time [min]

• 1.0e8

0.0e0 1.3e8 2.5e8 3.8e8 5.0e8 6.3e8 7.5e8 8.8e8 1.0e9 Intensity [counts]

RT: 2.10 RT: 2.56 RT: 3.19 RT: 4.10 RT: 4.48 RT: 5.16 RT: 5.62 RT: 6.49 RT: 12.84 RT: 12.92 RT: 13.31 RT: 13.89 RT: 14.12 RT: 14.47 RT: 14.96 RT: 15.13 RT: 15.26 RT: 15.36 RT: 15.55 RT: 15.68 RT: 15.97 RT: 16.09 RT: 16.28 RT: 16.33 RT: 16.40 RT: 16.51 RT: 16.67 RT: 17.09 RT: 17.22 RT: 17.41 RT: 17.47 RT: 17.76 RT: 17.84 RT: 18.03 RT: 18.25 RT: 18.43 RT: 19.18 RT: 19.76 RT: 20.63 RT: 20.85 RT: 21.05 RT: 21.24 RT: 21.73 RT: 21.78 RT: 22.18 RT: 22.28 RT: 22.51 RT: 22.83 RT: 22.99 RT: 23.23 RT: 23.29 RT: 23.46 RT: 23.66 RT: 24.25 RT: 24.29 RT: 24.63 RT: 27.18 RT: 27.29 RT: 27.34

min counts

PY-DSS_170712 #9 EPS321F_PY_2 TIC TIC

• Pentane
• Toluene
• Styrene
• Ethyl benzene
• Methyl styrene
• EMDP
• (2,3-diphenylcyclopropyl) methyl phenyl sulfoxide ,trans

¼Å¡ Ò ÃÇÔ à ¤ ÃÒ ÐËì à Á× Í à · Õ Âº ¡ Ñ º ° Ò ¹ ¢é Í ÁÙ Å´ é Ò ¹ ¾Í ÁÔ à ÁÍ Ã ì ¼ è Ò ¹ «Í ¿ áÇà ì F-Search

• Rank.2 : Acrylonitrile-Butadiene-Styrene copolymer ; ABS (C1-C40) Qual.81
• Rank.3 : Styrene-butadiene copolymer ABA block, 85% styrene (C1-C40) Qual. 81

ผลการวิเคราะห์ตัวอย่างที่ 4

0.1 5.0 10.0 15.0 20.0 25.0 30.0 31.1 Time [min]

• 1.0e8

0.0e0 1.0e8 2.0e8 3.0e8 4.0e8 5.0e8 6.0e8 7.0e8 8.0e8 Intensity [counts]

RT: 2.11 RT: 2.15 RT: 3.20 RT: 4.47 RT: 5.61 RT: 6.61 RT: 7.71 RT: 8.35 RT: 9.88RT: 10.49 RT: 11.56 RT: 11.71 RT: 11.86 RT: 11.95 RT: 12.83 RT: 12.91 RT: 12.97 RT: 13.40 RT: 14.19 RT: 14.47 RT: 15.12 RT: 15.23 RT: 15.36 RT: 15.96 RT: 16.00 RT: 16.10 RT: 16.22 RT: 16.33 RT: 16.45 RT: 16.67 RT: 17.08 RT: 17.48 RT: 17.72 RT: 17.81 RT: 17.90 RT: 18.69 RT: 18.83 RT: 18.90 RT: 19.19 RT: 19.38 RT: 19.60 RT: 19.82 RT: 20.29 RT: 21.00 RT: 21.19 RT: 22.14 RT: 22.55 RT: 23.08 RT: 23.29 RT: 23.37 RT: 23.50 RT: 23.62 RT: 26.35 RT: 27.26

min counts

PY-DSS_170712 #10 SANROPC_PY_2 TIC TIC

• Styrene
• Toluene
• 2-propenenitrile
• EMDP

¼Å¡ Ò ÃÇÔ à ¤ ÃÒ ÐËì à Á× Í à · Õ Âº ¡ Ñ º ° Ò ¹ ¢é Í ÁÙ Å´ é Ò ¹ ¾Í ÁÔ à ÁÍ Ã ì ¼ è Ò ¹ «Í ¿ áÇà ì F-Search

• Rank.2 : Acrylonitrile-Butadiene-Styrene copolymer ; ABS (C1-C40) Qual.79
• Rank.3 : Acrylonitrile styrene copolymer ; AS (C1-C40) Qual.76

¡ Ò Ã »Ã Ð ÂØ ¡ µì ã ª é PY-GCMS

88

• UV
• O2, H2O
• 1: Characterization of polymers
• 2: Quality control
• 3: Degradation/life evaluation of
• polymeric materials
• 4: Recycling of polymeric
• materials, biomass utilization
• 5: Organic geochemistry
• and soil chemistry
• 6: Clinical science, pathology
• 7: Biochemistry, microbiology
• 8: Coal liquefaction,
• energy conservation
• 9: Forensic science
• 10: Wood science,
• pulp industry
• 11: Tobacco smoke,
• toxicology
• 12: Extraterrestrial science
• 13: Environmental science

Your Scientific Specialist

Analysis PAHs in extender oils

90

Topics to be discussed

• Introduction PAHs
• Sample Preparation
• GCMSMS method
• Analysis PAHs
• LOD&LOQ
• Example of sample result
• Comment

91

Introduction

• Polycyclic aromatic hydrocarbons (PAHs) in extender oils and tyres are

produced using extender oils that may contain PAHs not added intentionally.

• PAHs are considered as toxic substances classified according to

Directive 67/548/EEC as carcinogenic, mutagenic and toxic for reproduction.

92

Scope for analysis.

• EU standard specifies a procedure for determination of benzo(a)pyrene

and sum of the eight individual polyaromatic hydrocarbons in extender

• ils. listed in Table1
• Sample Preparation Method : BS EN 16143:2013

Name of PAH Abbreviation CAS Registry number Benzo(a)pyrene BaP 50-32-8 Benzo(e)pyrene BeP 192-97-2 Benzo(a)anthracene BaA 56-55-3 Chrysene CHR 218-01-9 Benzo(b)fluoranthene BbFA 205-99-2 Benzo(j)fluoranthene BjFA 205-82-3 Benzo(k)fluoranthene BkFA 207-08-9 Dibenzo(a,h)anthracene DBahA 53-70-3

Table 1- List of individual PAHs in extender oils

93

PAHs... Consists of 8 natives of PAHs

MW range 228-278 amu (16PAHs could be up to 300+)

Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(j)fluoranthene Benzo(a)pyrene Benzo(e)pyrene Dibenzo(a,h)anthracene

C18H12

• MW. 228 g/mol

C22H14

• MW. 278 g/mol

C20H12

• MW. 252 g/mol

C20H12

• MW. 252 g/mol

94

Sample Preparation Process

(1) Prepares sample solution

Weight Sample 70 ± 0.1 mg into Vol. flask 5 ml Dissolve with 2 ml of n-Pentane and Spike internal Std. (deuterated IS)

(2) Deactivates silica

Deactivate Silica gel by stirring with 7% (m/m) of water for 24 h. 3.2 Load silica gel into chromatographic column (16 cm. L X 1 cm. ID)* 3.3 Flush silica gel with 10 ml n-Pentane through the column (discard) 3.4 Load sample (1) into column (before n-Pentane vanish form silica gel surface). 3.6 Elute sample by Cyclohexane 75 ml (several portion) and collect the eluents.** 3.7 Evaporate eluent (3.6) under 35 C till final volume 1ml. 3.1 Mix deactivated silica (in 2) 5 g with n-Pentane

(3) 1st sample extraction (8 Hours) Pack column Extracting

3.5 Rinse sample container with 2 ml n-Pentane.(not critical) and pour into column. *extended lenth of column to 25 cm. convenient for sample loading ** pressurized with N2 (1 bar est.) for faster elution

95

Sample Preparation Process

(4) Sample clean up (Sephadex LH20) (6 hours)

4.1 Mix 5 g. of Sephadex with IPA .. leave for overnight. 4.2 Load Sephadex into chromatographic column (12 cm L X 2.3 cm ID) 4.4 Rinse sample vessel with IPA (1 ml) and load into column. 4.7 Evaporate eluent (4.6) under 35 C till nearly dry. 4.8 Add 2 ml Acetone and evaporate till dry. 4.9 Dissolve with CycloC6 and transfer into 1 ml Vol.Flask 4.11 Make up volume to 1 ml wth Cyclohexane. 4.6 Collect eluent portion (@24-70 ml) in drying vessel

Fraction collecting Dissolved Solution

4.10 Add injection standard (DE)* 0.2 ml and make up volume to 1 ml with CycloC6 4.3 Add 1 ml IPA into (3.7) and load into column. 4.5 Elute with IPA at 1 ml/min, Discard the first 24 ml eluent. 4.12 Analyze with GCMSMS.

*DE = Decafluorodiphenyl

96

Instrument Method

GC parameters Parameter Value GC-column 60 m x 0.25 mm ID x 0.25 µm Stationary phase 17% phenyl-methylpolysiloxane Temperature program Initial 90 °C hold 1min 20°C /min to 250 °C 4°C /min to 330 °C hold 10 min Injection PTV, Splitless Injection temperature 275 °C Injection Volume 1 µL Carrier gas He UHP grade 1.2 ml/min

97

• Mass Spectrometer : EI – Temp 250 C/ TL Temp 330/
• MSMS – SRM Q1 resolution 0.7 FWHM, Q3 Resolution 0.7 FWHM

Instrument Method

Component RT mass product mass Collision energy

Decfluorodiphynly 5.84 333.9 233.9 35 333.9 264.9 25 Benzo(a)antracene-D12 18.46 240.1 212.1 25 240.1 236 30 Benzo(a)antracene 18.53 228.1 202 25 228.1 226 30 Chrysene 18.77 228.1 202 25 228.1 226 30 Benzo(b)Fluoranthene-D12 22.02 264.1 236 30 264.1 260 35 Benzo(b)fluoranthene 22.13 252.1 226 25 252.1 250 30 Benzo(k)fluoranthene 22.22 252.1 226.1 25 252.1 250 35 Benzo(j)fluoranthene 22.36 252.1 226 25 252.1 250 30 Benzo(e)pyrene 23.78 252.1 226.1 30 251.1 250 30 Benzo(a)pyrene-D12 23.89 264.2 236.1 30 264.2 260 35 Benzo(a)pyrene 24.03 252.1 226.1 35 251.1 250 30 Dibenzo(a,h)anthracene 30.23 278.1 276 35 278.1 276.2 50

98

8 PAHs Standard

1 3 2

TIC

99

Chromatogram (1) –Standard 8 PAHs with 3 IS(d12)

Benzo(a)anthracene-d12 Benzo(a)anthracene Chrysene

100

Chromatogram (2) –Standard 8 PAHs with 3 IS(d12)

Benzo(b)fluoranthene-d12 Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(j)fluoranthene

101

Chromatogram (3) –Standard 8 PAHs with 3 IS(d12)

Benzo(a)pyrene-d12 Benzo(e)pyrene Benzo(a)pyrene Tribenzo(a,h)anthracene

102

• Calculated from 10 replicate runs of TDAE sample (Treated Distillate Aromatic Extracted)

LOD/LOQ

8 compounds of PAHs have LOQ less than 0.1 mg/kg

Benzo(a)anthracene Chrysene Benzo(b)fluoranthene Benzo(k)fluoranthene Benzo(j)fluoranthene Benzo(e)pylene Benzo(a)pylene Dibenzo(a,h)anthracene

1

0.226 0.370 0.198 0.186 0.103

• 0.507

0.144 0.125

2

0.220 0.367 0.177 0.165 0.117

• 0.510

0.130 0.148

3

0.222 0.361 0.184 0.182 0.127

• 0.507

0.137 0.124

4

0.236 0.375 0.194 0.178 0.136

• 0.511

0.147 0.149

5

0.221 0.372 0.204 0.168 0.118

• 0.518

0.129 0.150

6

0.224 0.366 0.189 0.180 0.117

• 0.510

0.129 0.142

7

0.236 0.363 0.192 0.194 0.123

• 0.535

0.122 0.139

8

0.221 0.368 0.204 0.178 0.133

• 0.509

0.126 0.135

9

0.247 0.369 0.181 0.166 0.118

• 0.509

0.125 0.144

10

0.231 0.362 0.202 0.169 0.130

• 0.507

0.115 0.147

SD

0.0089 0.0045 0.0097 0.0095 0.0097 0.0086 0.0098 0.0095

LOD

0.0267 0.0134 0.0291 0.0285 0.0291 0.0258 0.0294 0.0286

LOQ

0.0891 0.0447 0.0969 0.0951 0.0972 0.0860 0.0980 0.0955

No.

PAHs (mg/ kg)

103

Peak Confirmation

Benzo(a)anthracene Chrysene

QC Check Sample spiked 3 ul of 0.5 mg/kg Sample(TDAE)

Chrysene Triphenylene

Benzo(b)fluoranthene Benzo(k)fluoranthene

104

Peak Confirmation

QC Check Sample spiked 3 ul of 0.5 mg/kg Sample-TDAE

Benzo(j)fluoranthene Benzo(e)pyrene Benzo(a)pyrene Dibenzo(a,h)anthracene

105

Result.. Recovery

• Two batches of analysis (2 replicates for each batch) from same sample (RPO)
• Recovery of PAHs : Deuterated IS vs. Injection Standard (Decafluorodiphenyl)
• BIU acceptable recovery is between 50% and 150%

Internal standard

Standard amount (mg)

Sample

Calculated amount (mg)

Acceptable Criteria

• f %Recovery

Verified

RPO_V1_Re01 RPO_V1_Re02

%Recovery

Benzo(a)anthracene-d12

4008

4663.572 4719.434

4691.503 117.05 (50-150) Pass

Benzo(b)fluoranthene-d12

4216

5684.548 5493.625

5589.087 132.57 (50-150) Pass

Benzo(a)pyrene-d12

4060

5389.764 5301.968

5345.866 131.67 (50-150) Pass

RPO_V2_Re01 RPO_V2_Re02 Benzo(a)anthracene-d12

4008

3532.543 3532.543

3532.543 88.14 (50-150) Pass

Benzo(b)fluoranthene-d12

4216

3249.254 3249.254

3249.254 77.07 (50-150) Pass

Benzo(a)pyrene-d12

4060

3319.878 3319.878

3319.878 81.77 (50-150) Pass

106

Comments

• Complicated & time consuming sample preparation – requires skills and

prone to error

• Improvement in separation (triphenylene vs. chrysene) can be done

upon availability of standard (triphenylene).

• Comparison study of purification between the two steps i.e. Silica Gel
• vs. Silica Gel & Sephadex are not so much different.
• New development on sample prep in order to reduce work loads and

improve analysis result.

Q&A